Advancements in medical research have led to unprecedented characterization of a plethora of factors involved in the development and progression of many diseases on cellular, subcellular, and molecular levels. Using advanced measurement techniques such as reverse transcriptase polymerase chain reaction (RT-PCR), advanced in vitro flow cytometry, and microchip fluid technology, researchers have gained valuable insights with potential applications in the treatment of many diseases.
A particularly challenging issue in medical research is the detection and characterization of relatively rare cells such as tumor-initiating cancer stem cells (CSCs), a treatment-resistant subclass of tumor cells thought to be responsible for the growth and regrowth of primary and metastatic tumors. The development of metastatic disease may be governed by the migration of CSCs from an existing tumor mass to remote locations via the circulatory or lymphatic system. The circulating tumor cells (CTCs) migrating within the circulatory or lymphatic systems typically include a relatively large number of differentiated tumor cells, and a tiny subset of undifferentiated stem circulating tumor cells (stem CTCs) associated with metastatic disease. Because of the rarity of the stem CTCs in circulation, their detection is exceedingly difficult due to limitations of existing measurement methods.
The sensitivity threshold of existing stem CTC assays, such as reverse transcriptase polymerase chain reaction (RT-PCR) assays, CellSearch system technology, in vitro flow cytometry, and microchip fluid technology is limited to approximately 1 cell per mL of sample due to the relatively small volume processed by the assay's instrumentation, which is typically about 10 mL or less. This sensitivity is inadequate to reliably detect these rare stem CTCs, which may circulate in concentrations of about 1 cell per 50 mL of blood or at even lower concentrations.
In vivo flow cytometry techniques, which detect cells as they circulate within the blood vessels of a living subject, overcome the sample volume limitation, since these techniques are capable of processing the entire blood volume of the subject as it passes through a particular blood vessel. However, for the detection of rare cells such as stem CTCs, the cell must pass though the area of interest of the instrumentation, typically a superficial blood vessel or lymph vessel. The amount of time required to observe a large volume of blood passing through the area of interest may be significant, especially when detecting and characterizing both the undifferentiated stem CTCs and the differentiated CTCs.
A need exists in the art to selectively enrich the concentration of cells circulating in vivo in order to enhance the sensitivity of the detection using in vivo flow cytometry techniques, among other needs. Such an enhancement would make possible the detection of relatively rare cells in a shorter amount of time, significantly increasing the cell detection sensitivity of the in vivo flow cytometry instrumentation. The increased cell detection sensitivity may allow for the earlier detection and treatment of diseases related to circulating rare cells such as the development of metastatic diseases from circulating stem CTCs.